High-Fat Diet Augments Myocardial Inflammation and Cardiac Dysfunction in Arrhythmogenic Cardiomyopathy

Arrhythmogenic cardiomyopathy (ACM) is a familial heart disease characterized by cardiac dysfunction, arrhythmias, and myocardial inflammation. Exercise and stress can influence the disease’s progression. Thus, an investigation of whether a high-fat diet (HFD) contributes to ACM pathogenesis is warranted. In a robust ACM mouse model, 8-week-old Desmoglein-2 mutant (Dsg2mut/mut) mice were fed either an HFD or rodent chow for 8 weeks. Chow-fed wildtype (WT) mice served as controls. Echo- and electrocardiography images pre- and post-dietary intervention were obtained, and the lipid burden, inflammatory markers, and myocardial fibrosis were assessed at the study endpoint. HFD-fed Dsg2mut/mut mice showed numerous P-wave perturbations, reduced R-amplitude, left ventricle (LV) remodeling, and reduced ejection fraction (%LVEF). Notable elevations in plasma high-density lipoprotein (HDL) were observed, which correlated with the %LVEF. The myocardial inflammatory adipokines, adiponectin (AdipoQ) and fibroblast growth factor-1, were substantially elevated in HFD-fed Dsg2mut/mut mice, albeit no compounding effect was observed in cardiac fibrosis. The HFD not only potentiated cardiac dysfunction but additionally promoted adverse cardiac remodeling. Further investigation is warranted, particularly given elevated AdipoQ levels and the positive correlation of HDL with the %LVEF, which may suggest a protective effect. Altogether, the HFD worsened some, but not all, disease phenotypes in Dsg2mut/mut mice. Notwithstanding, diet may be a modifiable environmental factor in ACM disease progression.


Introduction
Arrhythmogenic cardiomyopathy (ACM) is an inherited, nonischemic heart disease (NIHD) characterized by ventricular dysfunction, arrhythmias, and myocardial inflammation and fibrosis [1].Despite its first description in the 1980-1990s [2,3], there is still no available drug to treat the underlying disease pathogenesis.ACM is often called "a disease of the cardiac desmosome" [4], as the first and second most prevalent pathogenic gene variants that give rise to ACM are the desmosomal genes Plakophilin-2 (PKP2) and Desmoglein-2 (DSG2) [5].Interestingly, PKP2 variants give rise to a right-ventriculardominant form of ACM, whereas DSG2 variants are left-ventricular dominant [6,7].ACM is considered a disease with incomplete penetrance and variable expressivity, and external factors (e.g., stress, anxiety, and exercise) have been known to influence this disease's manifestation and progression [4,[8][9][10][11][12].Whereas other lifestyle factors, such as diet, encompass an area that is largely unexplored in ACM progression and severity.On average, United States (US) adults consume a diet high in saturated fat, which is associated with arterial plaque formation, worsened systolic function, and elevated levels of circulating lipids [13][14][15][16].Specifically, triglycerides (TGs) and elevated circulating cholesterols, including very low-and low-density lipoproteins (VLDLs and LDLs, respectively), are risk factors for cardiovascular disease (CVD), while high-density lipoprotein (HDL) cholesterol is regarded as heart-healthy, largely due to its ability to shuttle cholesterol for biliary excretion [17].
Poor diet is linked to obesity, an epidemic linked to a myriad of CVDs through dyslipidemia and the promotion of inflammatory pathways [18].Given that fat is the most calorie-dense macronutrient, maintaining dietary fat within the recommended 20-35% daily consumption is of critical importance [19].Astoundingly, Americans consume a diet consisting of, on average, 36% fat per day [20].In addition, despite recommendations limiting saturated fat to 5-6% per day [19], the U.S. population consumes a diet enriched with fat far beyond the recommendations (11-12% per day) [21].
Saturated fat can elevate inflammatory cytokines [22,23], which were recently identified to drive myocardial injury, arrhythmias, and dysfunction in ACM [24,25].Occurring in three out of four ACM patients, myocardial inflammation is a causative phenotype linked to premature death [2,26,27].Recent work has demonstrated that the master regulator of inflammation and the innate immune response, NFκB, increases inflammatory cytokine and chemokine levels via the mobilization of immune cells to the hearts of ACM subjects [24,25].Thus, disease expressivity and severity can be influenced by intrinsic (i.e., myocyte-mediated NFκB chemotaxis), cardiac-extrinsic (i.e., circulating factors), and environmental (i.e., stress and exercise) stimuli.As such, the topic of whether a high-fat diet (HFD) can influence disease progression in this familial heart disease warrants investigation.
A previous study reported that the HFD induced certain disease phenotypes in a Plakophilin-2 heterozygous (Pkp2 +/− ) mouse (e.g., 10-week-old mice received an HFD for 3 months) [28].However, these mice were asymptomatic until an HFD was introduced, indicating that this environmental factor could be sufficient to induce disease onset in genotype-positive, phenotype-negative (G+/P−) ACM patients.Yet, whether an HFD is sufficient to influence disease progression in G+/P+ ACM subjects is unknown.Conversely, this body of work utilizes an ACM mouse model that develops cardiac dysfunction by early adolescence (<8 weeks old)-without exogenous stimuli (i.e., diet-induced).Considering elevated fat consumption in the general population and the cardiovascular effects of an HFD on healthy individuals as well as those with acquired heart diseases, we investigated the influence of an HFD in a preclinical animal model of ACM.In essence, whether an HFD exacerbates existing disease phenotypes (i.e., rather than inducing them [28]) has yet to be elucidated.

Animal Studies
Wildtype (WT) controls and homozygous Desmoglein-2 mutant (Dsg2 mut/mut ) mice [29] were enrolled in an 8-week dietary intervention study at 8 weeks of age.All the mice were housed in the Florida State University College of Medicine vivarium in a temperature-and humidity-controlled environment with a 12 h light-dark cycle with access to food and water ad libitum.All the experiments were approved under the Florida State University Animal Care and Use Committee (Protocol No. PROTO202000052).This study used both male and female mice, with equal sexes randomized to one of two diets: a purified HFD (60% kcal fat, 20% kcal carbohydrate, and 20% kcal protein; Research Diets, New Brunswick, NJ, USA, Cat.No. D12492) or standard rodent chow, non-purified diet (16.5% kcal fat, 56.8% kcal carbohydrate, 26.6% kcal protein; Lab Diets, Richmond, IN, USA, Cat.No. 5008).

Mouse Echo-and Electrocardiography
The cardiac function was assessed via echocardiography (Echo) using the Vevo F2 Visualsonic imaging system (FUJIFILM VisualSonics, Toronto, ON, Canada) with a UHF57x linear transducer (57-25 MHz) and 8-lead surface electrocardiography (ECG) using the iWork IX-BIO-SA Small Animal ECG System (Cat.No. TCS-100; iWorx Systems Inc., Dover, NH, USA).Echos and ECGs were performed on anesthetized mice (nose cone anesthesia, 1.5-2% isoflurane vaporized in 100% O 2 ) prior to dietary intervention (8 weeks of age) and at study endpoint (16 weeks of age).The Echos were performed, measured, and analyzed according to the American Society of Echocardiography guidelines [30].At least three shortaxis (M-mode) and parasternal long-axis (B-mode) videos were acquired for each mouse and averaged, as previously described [29].Five-minute ECG recordings were obtained via surface ECG telemetry electrodes following manufacturer's protocol (iWork, IX-BIO8) and analyzed via iWork LabScribe v24 software following the construction of signal-averaged ECGs (SAECGs) from five-minute recordings.Following endpoint functional studies, the mice were euthanized, and tissues were collected and either flash-frozen in liquid nitrogen or stored in 10% buffered formalin.The flash-frozen samples were transferred to −80 • C, and fixed samples were processed and embedded in paraffin blocks as described below.

Plasma Marker Assessment
Blood was collected in lavender EDTA-coated tubes (BD Microtainer, Franklin Lakes, NJ, USA; Cat.No. 365974) and centrifuged at 2000× g for 10 min at 4 • C: the plasma was then transferred to Eppendorf tubes and stored at −80 • C until downstream use.The plasma samples were diluted (1:2-1:4) in 1X PBS, and then, 100 µL of diluted plasma samples were individually added to Lipid Panel Plus Reagent Discs (McKesson, Irving, TX, USA; Cat.No. 07P0205) and read in a Chemistry Analyzer Piccolo Xpress (Abbott, Chicago, IL, USA).Due to the variations in the sample concentrations and the sensitivity of the Piccolo Xpress analyzer, certain samples were re-run at different dilutions (i.e., 1:2-1:4) to be accurately assessed.Each sample was read individually using disc-specific calibration data via individual bar codes printed on each disc.Additionally, even when a sample is read by the machine, the output may not provide values for all markers.This is often due to undetectable levels of certain types of cholesterol.

Cardiac Cytokine Arrays
Frozen hearts were lysed in a RIPA buffer (ThermoFisher, Waltham, MA, USA; Cat.No. 89900) with 1:100 protease (Sigma-Aldrich, Cat.No. P8430) and phosphatase inhibitor cocktails (Sigma-Aldrich, Cat.No. P0044).The protein levels were standardized using a Pierce BCA protein assay kit (ThermoFisher, Cat.No. 23227).Mouse Proteome Profiler XL Cytokine Arrays (R&D Systems, Minneapolis, MN, USA; Cat.No. ARY028) were blocked for 1 h at room temperature before the membranes were incubated at 4 • C with 200 µg of protein overnight.The following day, the membranes underwent the following steps with three water washes (10 min each) between each step: (i) incubated for 1 h at room temperature with antibody detection cocktail, (ii) incubated with 1:2000 streptavidin horseradish peroxidase for 30 min at room temperature, and then (iii) SuperSignal West Pico PLUS chemiluminescence substrate (ThermoFisher, Cat.No. 34577) was added before imaging on an Azure Biosystems 400 imager (Azure Biosystems Inc., Dublin, CA, USA).The images were quantified using the Ideal Eyes Systems, Inc. (Bountiful, UT, USA) QuickSpots software (version 25.6.0.1).

Statistical Analysis
The data are presented as the mean ± SEM with p ≤ 0.05 considered statistically significant.Additional statistical information is included in each figure legend.GraphPad PRISM was used for all statistical computations and graph preparations.Differences between groups and treatments were assessed with one-and two-way ANOVAs with Tukey's post hoc analysis as appropriate, and normal distributions were assumed.Pearson's correlation coefficient (r) was used to determine correlations between the Echo parameters and the circulating lipids.

Results
3.1.Impaired Cardiac Function and ECG Anomalies Following High-Fat-Diet Exposure in Dsg2 mut/mut Mice As aforementioned, this study was designed to fill a gap in our knowledge of environmental factors that impact ACM, namely, how an HFD affects disease progression in ACM.The choice of HFD for this project was one of the most common HFDs, if not the most common HFD, for basic scientists.Sold for over 20 years by the largest supplier of rodent diets, Research Diets, the 60% kcal fat diet reduces the time of animal housing and the study duration without sacrificing study quality [31].Specifically, in comparison to the 45% kcal fat diet offered by Research Diets, the metabolic response differences are very small [31].
Thus, during this dietary modification period, which occurred from 8 to 16 weeks of age, the Dsg2 mut/mut mice received standard rodent chow or an HFD (60% kcal fat).WT mice maintained on the same standard rodent chow served as the study control group.As expected, the HFD increased body weight of the Dsg2 mut/mut mice, as seen in Figure S1A.
The most notable changes in cardiac function are portrayed in Figure 1, while additional echocardiographic data are compiled in Table 1.As assessed by the percent left ventricular ejection fraction (%LVEF), compared to WT controls (Figure 1A,B), cardiac dysfunction was similar in both Dsg2 mut/mut cohorts prior to dietary intervention.These data ensured uniformity prior to study initiation and enabled us to assess whether an HFD can exacerbate existing disease phenotypes.While both chow-and HFD-fed 16-week-old Dsg2 mut/mut mice continued to display reduced cardiac function (both p < 0.0001 compared to WT controls), further impairment in cardiac function was observed in HFD-fed 16-weekold Dsg2 mut/mut mice (p < 0.0011 compared to Dsg2 mut/mut mice fed chow; Figure 1A,B, Table 1).This finding indicated that exposure to a diet high in fat is sufficient to potentiate LV dysfunction in ACM mice.PR-I, PR interval; Pd, P-wave duration; QRSd, QRS duration; P-Amp, P-amplitude; R-Amp, R-amplitude; Q-Amp, Q-amplitude; S-Amp, S-amplitude.Data presented as mean ± SEM or percent J-wave depression (via mice that showed J-wave depression divided by total mice within their respective cohort × 100).n-values inset; * p ≤ 0.05 for any cohort vs. WT + chow; † p ≤ 0.05 for Dsg2 mut/mut + HFD vs. Dsg2 mut/mut + chow using one-way ANOVA with Tukey's post hoc.
A variety of ECG anomalies were identified at 16 weeks of age (Figure 1C, Table 1).Specifically, the P-wave duration was shortened in chow-(p = 0.0021) and HFD-fed (p = 0.0016) Dsg2 mut/mut mice compared to WT controls (Figure 1D,E).Additionally, the P-wave amplitude was reduced in chow-(p = 0.2361) and HFD-fed (p = 0.0043) Dsg2 mut/mut mice compared to WT controls (Figure 1D,E).Similar to our previous findings [24], the S-amplitude trended lower in Dsg2 mut/mut mice fed standard rodent chow, yet Dsg2 mut/mut fed an HFD displayed near similar values in S-amplitude compared to WT controls (Figure 1F).In addition, as observed in Figure 1C and Table 1, the R-amplitude was diminished in both Dsg2 mut/mut groups compared to the WT controls.In light of the further decline in cardiac function in HFD-fed Dsg2 mut/mut mice and considering that a reduced %EF is a major Task Force criteria for ACM [32], we performed correlation analyses between Echo parameters for this cohort (Figure 2A,B).The echocardiographic analyses demonstrated increased LV mass (LVM) with increased thinning of the LV wall (RWT; Figure 2A) in HFD-fed Dsg2 mut/mut mouse hearts.And a strong, positive correlation between %LVEF and RWT (r = 0.77) and %LVEF and LV fractional shortening (%LVFS, r = 1.0) was observed (Figure 2C,D).In short, the %LVEF, RWT, and %LVFS were all drastically lower in Dsg2 mut/mut mice fed an HFD than those in all cohorts (p < 0.01).Thus, our results demonstrate that an HFD not only led to further cardiac dysfunction but was an additional driver of cardiac remodeling.3.2.HFD-Induced Elevations in Plasma Lipids in Dsg2 mut/mut Mice Next, we measured plasma lipids and whether they correlated with cardiac functional parameters (Figure 3A) or with additional circulating factors (Figure 3B)-an investigative outcome that could lead to dietary recommendations in patients with ACM.The HFD elevated the circulating levels of total cholesterol (p < 0.001 compared to both cohorts, TC, Figure 3C).This finding was novel, as a previous report in mice with the same C57BL/6 background as the Dsg2 mut/mut mice had a trend toward a higher TC with 16 weeks of an HFD modification [33].The majority of the cholesterol was in LDL in HFD-fed Dsg2 mut/mut mice (Figure 3D), which was undetected in both cohorts on chow diet.However, the HDL was increased in HFD-fed Dsg2 mut/mut mice compared to WT mice (p < 0.0348) and Dsg2 mut/mut mice (p < 0.0060) fed chow (Figure 3E).Of interest, the only lipid that correlated with %LVEF was HDL (r = 0.83), indicating that the HDL may act to preserve cardiac function in this ACM model.Surprisingly, TG and VLDL, strong CVD risk factors [34], were elevated in Dsg2 mut/mut mice fed standard rodent chow but were similar between WT mice fed chow and Dsg2 mut/mut mice fed an HFD (Figure S1B,C).In line with the observed increase in LDL, the nHDL-C was additionally elevated in Dsg2 mut/mut mice fed an HFD, while both Dsg2 mut/mut cohorts displayed a higher TC/HDL ratio (Figure S1D,E).Lastly, the liver enzymes, ALT and AST, are both associated with organ-specific and systemic inflammation [7], yet they were unchanged between cohorts, as was glucose (Figure S1F-H).Overall, our plasma analyses indicated a paucity of correlation between circulating lipids and cardiac function.Lastly, and most surprisingly, it is worth repeating that a potential cardioprotective mechanism involves circulating HDL levels in response to an elevated dietary fat consumption in ACM mice.

HFD-Induced Elevations in Plasma Lipids in Dsg2 mut/mut Mice
Next, we measured plasma lipids and whether they correlated with cardiac functional parameters (Figure 3A) or with additional circulating factors (Figure 3B)-an investigative outcome that could lead to dietary recommendations in patients with ACM.The HFD elevated the circulating levels of total cholesterol (p < 0.001 compared to both cohorts, TC, Figure 3C).This finding was novel, as a previous report in mice with the same C57BL/6 background as the Dsg2 mut/mut mice had a trend toward a higher TC with 16 weeks of an HFD modification [33].The majority of the cholesterol was in LDL in HFD-fed Dsg2 mut/mut mice (Figure 3D), which was undetected in both cohorts on chow diet.However, the HDL was increased in HFD-fed Dsg2 mut/mut mice compared to WT mice (p < 0.0348) and Dsg2 mut/mut mice (p < 0.0060) fed chow (Figure 3E).Of interest, the only lipid that correlated with %LVEF was HDL (r = 0.83), indicating that the HDL may act to preserve cardiac function in this ACM model.Surprisingly, TG and VLDL, strong CVD risk factors [34], were elevated in Dsg2 mut/mut mice fed standard rodent chow but were similar between WT mice fed chow and Dsg2 mut/mut mice fed an HFD (Figure S1B,C).In line with the observed increase in LDL, the nHDL-C was additionally elevated in Dsg2 mut/mut mice fed an HFD, while both Dsg2 mut/mut cohorts displayed a higher TC/HDL ratio (Figure

HFD Consuption Elevated Myocardial Inflammatory Cytokines in Dsg2 mut/mut Mice
Continuing our investigation into potential instigators that could be responsible for the adverse effect on cardiac function in HFD-fed Dsg2 mut/mut mice, we assessed myocardial fibrosis (Figure 4A).As previously reported by our group, healthy, age-matched WT hearts have null-to-minimal fibrosis, while Dsg2 mut/mut mice at 16 weeks have severe fibrotic scarring [24].Although similar findings were noted in chow-fed Dsg2 mut/mut mice compared to WT controls, our results indicated that the HFD did not have a compounding effect on cardiac fibrosis (Figure 4A,B).
S1D,E).Lastly, the liver enzymes, ALT and AST, are both associated with organ-specific and systemic inflammation [7], yet they were unchanged between cohorts, as was glucose (Figure S1F-H).Overall, our plasma analyses indicated a paucity of correlation between circulating lipids and cardiac function.Lastly, and most surprisingly, it is worth repeating that a potential cardioprotective mechanism involves circulating HDL levels in response to an elevated dietary fat consumption in ACM mice.Since the fibrotic burden was not elevated in response to this dietary modification, we turned our attention to cardiac inflammation, a key pathological signature of ACM [2,26,27] that has been previously reported in this mouse model [24,25].We found numerous markers of cardiac inflammation that were elevated with an HFD compared to Dsg2 mut/mut and WT mice fed chow (Figures 4C,D, S2, and S3).Specifically, in Dsg2 mut/mut Since the fibrotic burden was not elevated in response to this dietary modification, we turned our attention to cardiac inflammation, a key pathological signature of ACM [2,26,27] that has been previously reported in this mouse model [24,25].We found numerous markers of cardiac inflammation that were elevated with an HFD compared to Dsg2 mut/mut and WT mice fed chow (Figures 4C,D and S2 and S3).Specifically, in Dsg2 mut/mut mice fed chow, cardiac elevations in periostin (POSTN; two-to threefold), tumor necrosis factor alpha (TNFα; three-to fourfold), and pentraxin-2 (PTX2; three-to fourfold) and -3 (PTX3; two-to threefold) were observed (Figures S2 and S3A) compared to chow-fed WT mice.When HFD-fed Dsg2 mut/mut mice were compared against WT controls, matrix metalloproteinases-2/3/9 (MMP2, two-to threefold; MMP3, three-to fourfold; and MMP9, three-to fourfold; respectively), osteopontin (OPN, two-to threefold), lipocalin-2 (LCN2, two-to threefold), plasminogen activator inhibitor-1 (PAI-1, three-to fourfold), myeloperoxidase (MPO, two-to threefold), resistin (RETN, two-to threefold), C-X-3C motif chemokine ligand-1 (CX3CL1, two-to threefold), C-C motif chemokine ligand-21 (CCL21; two-to threefold), and PTX2 (two-to threefold) were all elevated (Figures S2 and S3B).Thus, a myriad of myocardial inflammatory markers were elevated in response to ACM mice fed an HFD (Figures S2 and S3C).
Lastly, fold changes do not take into account the total protein levels of inflammatory markers, as these are normalized to WT controls.Thus, we additionally assayed these markers as picograms per milliliter (pg/mL), which highlighted even more findings (presented in Figure 5).This additional analysis was performed considering the physiological role and relevance that each cytokine and/or chemokine imparts (Figure 5).Thus, the inflammatory markers were categorized into their respective functional role, as previously described [35].For cytokine ligands, adiponectin (AdipoQ) was the only marker elevated in cardiac tissue from Dsg2 mut/mut mice fed an HFD compared to both Dsg2 mut/mut and WT mice fed a chow diet (Figure 5A).When grouped by their role in chemotaxis, the most elevated levels of chemokines were observed for CCL21 and CX3CL1 in Dsg2 mut/mut mice fed an HFD compared to both chow-fed cohorts (Figure 5B).The inflammatory receptors and those involved in cascade signaling, CD93 and soluble ICAM-1 (sICAM-1), were elevated in HFD-fed Dsg2 mut/mut mice compared to the other two cohorts (Figure 5C).Additionally, HFD-fed Dsg2 mut/mut mice harbored higher levels of four markers involved in enzymatic activity, including cluster of differentiation 142 (CD142, aka thromboplastin), C-reactive protein (CRP), PTX2, and PAI-1 (Figure 5D).Within the hormone and growth factor group, Dsg2 mut/mut mice fed an HFD (compared to Dsg2 mut/mut and WT mice fed chow) showed the highest levels of fibroblast growth factor-1 (FGF1; >18,000 pg/mL) compared to any other cytokine (Figure 5E,F), an unexpected finding.Lastly, compared to both chow-fed cohorts, Dsg2 mut/mut mice fed an HFD had potentiated levels of several markers, including fetuin A (AHSG), endoglin (CD105), and endostatin (Figure 5F).All of these inflammatory markers are known to augment cardiac remodeling and can additionally alter electrophysiological conduction via autocrine/paracrine signaling of neighboring ion channels [36,37].

HFD and Cholesterol: Their Role in Myocardial Inflammation
As previously described, in a complex and intricate network of signaling events, an HFD elicits multiorgan changes, driving systemic inflammation [38].The HFD elevates endotoxins and free fatty acids, increases NFκB signaling and oxidative stress, and triggers the activation of M1 macrophages [38].This leads to the production of an abundance of inflammatory cytokines that can induce systemic inflammation and promote a myriad of diseases [38].In atherosclerosis and coronary heart disease, an HFD elevates IL-6, Creactive protein (CRP), and TNF-α preceding endothelial dysfunction and nitric oxide depletion.The HFD's role in nonischemic heart diseases, such as ACM, is largely unknown, particularly regarding whether it can exacerbate existing ACM phenotypes.Considering prior studies demonstrating the role of NFκB signaling and oxidative stress in ACM [12,24], we suspected that an HFD would augment these existing phenotypes.
The HFD upregulated the TC, most of which was LDL, while the HDL was also increased.A higher baseline HDL and undetectable levels of LDL with the chow diet were expected, as mice preferentially carry cholesterol on HDL in contrast to humans, who preferentially carry cholesterol on LDL [39].As such, LDL has long been considered a risk factor and therapeutic target for CVDs [40].Furthermore, elevated levels of oxidized LDL (oxLDL) have previously been observed in ACM patients and were associated with cardiac dysfunction, structural remodeling, and ECG abnormalities [28].Through additional sophisticated iPSC and animal studies, Sommariva E. and colleagues demonstrated that these phenotypes were the result of PPARγ-dependent cardiac adipogenesis [28], an additional pathway implicated by us [41].
Here, the positive association of HDL with the %LVEF in ACM mice fed an HFD led us to believe that HDL may exert protective effects.Widely regarded for its role in reverse cholesterol transport, HDL also has anti-oxidant and anti-apoptotic effects [17].However, most recently, the oxidation of HDL has been associated with atrial fibrillation (AFib) [42], while high HDL has been associated with dementia (>90 mg/dL) [43] and, in male patients, with hypertension, a CV risk factor (>80 mg/dL) [44].Further investigation is warranted, especially considering the disparate outcomes in human studies, where HDL showed a U-shaped association, in which low and high levels are associated with poor outcomes [45].In particular, high levels of HDL either showed no cardiovascular benefits [45] or were associated with increased CVD-related mortality at severely elevated levels, such as ≥90 mg/dL [46] or ≥116 mg/dL [47], from two independent studies.Overall, attention to the oxidation status and plasma levels in relation to other health indices is vital to delineate the protective or adverse effects of HDL cholesterol.

Inflammation-Induced Cardiac Remodeling
CD93 is primarily expressed by endothelial cells, which line the endocardium [48], thus making this a suitable environment for CD93 storage and release.Additionally, elevated levels of circulating CD93 are associated with cardiac mortality and AFib [49].CD93 and sICAM-1 were elevated in HFD-fed Dsg2 mut/mut mice compared against the other two cohorts (threefold and twofold, respectively).Considering that both CD93 and sICAM-1 are known promoters of atherosclerosis, it is not surprising that these inflammatory markers would additionally compound existing CVDs [50,51].In addition, in mice with heart failure, sICAM-1 mediates leukocyte infiltration, leading to LV cardiac remodeling and subsequent dysfunction and fibrosis [52].Conversely, we saw cardiac dysfunction in the absence of heightened fibrosis in HFD-fed Dsg2 mut/mut mice.This could be a consequence of MMP overexpression.CRP levels were also elevated with an HFD in Dsg2 mut/mut mice.CRP is secreted by immune cells and ACM myocytes in response to acute inflammation [53], and decreased levels of CRP are associated with QT shortening [54].It did come as a surprise to see elevated PXT2 levels with HFD, as this cytokine is involved in antifibrotic and anti-inflammatory signaling pathways [55].However, PXT2 was not associated with other HFD-induced upregulated cytokines in ACM subjects, as indicated in Supplementary Figure S2C.Thus, its increase may be a protective mechanism against myocardial fibrosis.
Alternatively, it comes as no surprise that AdipoQ was high in all mice compared to other markers, as this circulating hormone is the most abundant adipokine in circulation [56], with both atheroprotective and atheropromoting effects reported by us previously [57].Observationally, increased plasma levels are associated with a heightened risk of heart failure, AFib, aortic valve stenosis, and myocardial infarction (MI) [58], while another study found that reduced AdipoQ levels were associated with AFib [59].While AdipoQ can be secreted by a wide array of cell types, including aortic vascular smooth muscle cells [60], the primary cell type that secretes this adipokine are adipocytes [61].Here, the observation of elevated myocardial levels of AdipoQ in response to the HFD suggests that this adipokine may be involved in ACM fibrotic scarring and/or adipogenesis, hallmarks of ACM pathology [62,63].Overall, while these inflammatory marker elevations do not appear to further myocardial fibrosis, they may be involved in cardiac remodeling.
A reduced level of fetuin-A is linked to LV remodeling, increased mortality, post-MI, and ECG alterations, including incomplete ST segment resolution [64].Thus, elevated AHSG with the HFD in our ACM mouse model may be protective.COL18A1, which was elevated with the HFD, has been associated with right ventricle dysfunction [65].PAI-1, a coagulation factor increased with an HFD, is associated with prevalent and incident AFib [66].CCL21 is also associated with detrimental effects including associations with all-cause mortality, myocardial inflammation and fibrosis, and chronic heart failure [67,68].Lastly, the prevention of CCL21 binding to CCR2 prevents myocyte action potential (AP) prolongation [69].
CX3CL1 was an additional inflammatory marker elevated by the HFD.CX3CL1 (aka, fractalkine) is associated with AFib [70], promotes leukocyte recruitment and monocyteadipocyte adhesion [71], and upregulates MMPs and ICAM [72].CX3CL1 affects numerous cell-signaling pathways, including NFκB and Wnt/β-catenin [73].As P-wave abnormalities are indicative of the established involvement of canonical Wnt signaling in the control of AV junction electrophysiology [74], it is conceivable that CX3CL1 could contribute to P-wave alterations.
Also increased by the HFD was CD105, which has both anti-and profibrotic effects [75].When CD105 activity is blunted, cardiac fibrosis is also blunted and associated with reduced heart failure mortality [76].CD105 is a member of the transforming growth factor-β (TGFβ) receptor complex and promotes endothelial cell proliferation and migration [77].On the other hand, CD105 downregulates TGF-β and NFκB signaling to exert antifibrotic effects [78].Thus, our findings may implicate elevated CD105 levels as an antifibrotic compensatory mechanism as a potential cardioprotective cytokine.
FGF1 levels were substantially increased in Dsg2 mut/mut mice fed an HFD; in fact, FGF1 was the most elevated cytokine in this cohort.Given FGF1 is involved in fibroblast and endothelial cell growth during cardiac cell repair [79], it affords a plausible explanation for the stark potentiation of FGF1.Also elevated in mice subjected to an HFD, RETN is secreted by adipocytes, promoting inflammation in an NFκB-dependent mechanism [80].This is particularly interesting, considering NFκB signaling is implicated in ACM pathogenesis and could serve as a therapeutic target in the treatment of ACM [24,25,81].Increased MMPs (-2, -3, and -9) suggest extracellular matrix (ECM) remodeling and fibrosis, key ACM cardiac alterations, which can be further investigated in vivo and in vitro.While we did not see increased cardiac fibrosis in HFD-fed Dsg2 mut/mut mice, it is worth noting that MMP-induced ECM remodeling increases vascular permeability and, thus, access to infiltrating immune cells in ACM hearts [82], a finding that we recently reported was the result of infiltrating CCR2 + macrophages [25].

HFD-Induced ECG Alterations and Cardiac Dysfunction
Although the P-amplitude was reduced in both Dsg2 mut/mut cohorts, only those mice on the HFD showed a significant reduction compared to WT controls.This is particularly interesting given that our prior studies demonstrated no difference in the P-amplitude between WT and Dsg2 mut/mut mice [24,29], except in exercised Dsg2 mut/mut mice [12].Thus, demonstrating that both environmental factors (i.e., diet and exercise) can influence atrial depolarization.
Although originally termed arrhythmogenic right ventricular cardiomyopathy (ARVC), considering the prevalence of right-ventricular-dominant phenotypes, now this dreadful disease can also manifest as biventricular disease or as left-dominant ACM [83].Leftdominant disease is more prevalent in subjects harboring a DSG2 pathogenic variant; and thus, in addition to the observed reductions in the %LVEF in HFD-fed Dsg2 mut/mut mice, there were other LV echocardiographic indices worth noting.In particular, Dsg2 mut/mut + HFD mice showed increased thinning of the LV wall (i.e., relative wall thickness [RWT]), in conjunction with increased LV mass (LVM) and severely reduced LV fractional shortening (%LVFS; Table 1).Both indices, RWT and LVM, were far worse compared to WT + chow as well as HFD-fed Dsg2 mut/mut mice (Table 1).The further and stark decline in the %LVFS in HFD-fed Dsg2 mut/mut mice compared to all cohorts is of clinical importance, given that LV dysfunction is traditionally defined as having a %LVEF of less than 50% and a %LVFS of less than 28% [84,85] in patients with heart disease.However, our findings indicate that the %LVFS may be a diet-induced functional parameter worth monitoring in ACM subjects, given the fact that HFD-fed Dsg2 mut/mut mice failed to achieve an LVFS of 28% or more.

Conclusions
Originally believed to be a congenital heart disease (i.e., ARVD), five decades of research has now uncovered that ACM is a progressive and primary NIHD arising from, chiefly, pathogenic desmosomal variants.Environmental Factors: Numerous studies have demonstrated the following: (i) Exercise worsens disease progression and can induce fatal arrhythmias, yet only secondary to the underlying antecedent (i.e., pathogenic variant).(ii) Recent work even demonstrated the impact of psychosocial stress on potentiating the arrhythmic burden in both mouse models of ACM and patients with ACM.Thus, (iii) whether an HFD could exacerbate disease phenotypes is of monumental importance, as this is a modifiable environmental factor.Our findings further advance the field of ACM, by demonstrating that an HFD further increases the circulating TC, potentiating cardiac dysfunction and myocardial inflammation, in the presence of the primary root cause (i.e., Dsg2-mutation).Thus, while an HFD is a well-known contributor to cholesterol burden, atherosclerosis, and CVDs, it is not the precursory trigger in cardiac dysfunction in ACM mice.However, it should be considered an environmental factor that advances disease progression.